Nanosecond Transient IR Spectroscopy of Halorhodopsin in Living Cells

The ability to track minute changes of a single amino acid residue in a cellular environment is causing a paradigm shift in the attempt to fully understand the responses of biomolecules that are highly sensitive to their environment. Detecting early protein dynamics in living cells is crucial to understanding their mechanisms, such as those of photosynthetic proteins. Here, we elucidate the light response of the microbial chloride pump NmHR from the marine bacterium Nonlabens marinus, located in the membrane of living Escherichia coli cells, using nanosecond time-resolved UV/vis and IR absorption spectroscopy over the time range from nanoseconds to seconds. Transient structural changes of the retinal cofactor and the surrounding apoprotein are recorded using light-induced time-resolved UV/vis and IR difference spectroscopy. Of particular note, we have resolved the kinetics of the transient deprotonation of a single cysteine residue during the photocycle of NmHR out of the manifold of molecular vibrations of the cells. These findings are of high general relevance, given the successful development of optogenetic tools from photoreceptors to interfere with enzymatic and neuronal pathways in living organisms using light pulses as a noninvasive trigger

The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis

Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis

Membrane Protein Activity Induces Specific Molecular Changes in Nanodiscs Monitored by FTIR Difference Spectroscopy

It is well known that lipids neighboring integral membrane proteins directly influence their function. The opposite effect is true as well, as membrane proteins undergo structural changes after activation and thus perturb the lipidic environment. Here, we studied the interaction between these molecular machines and the lipid bilayer by observing changes in the lipid vibrational bands via FTIR spectroscopy. Membrane proteins with different functionalities have been reconstituted into lipid nanodiscs: Microbial rhodopsins that act as light-activated ion pumps (the proton pumps NsXeR and UmRh1, and the chloride pump NmHR) or as sensors (NpSRII), as well as the electron-driven cytochrome c oxidase RsCcO. The effects of the structural changes on the surrounding lipid phase are compared to mechanically induced lateral tension exerted by the light-activatable lipid analogue AzoPC. With the help of isotopologues, we show that the ν(C = O) ester band of the glycerol backbone reports on changes in the lipids’ collective state induced by mechanical changes in the transmembrane proteins. The perturbation of the nanodisc lipids seems to involve their phase and/or packing state. 13C-labeling of the scaffold protein shows that its structure also responds to the mechanical expansion of the lipid bilayer

Ubiquitin protease Ubp8 is necessary for S. cerevisiae respiration

Healthy mitochondria are required in cell metabolism and deregulation of underlying mechanisms is often involved in human diseases and neurological disorders. Post-translational modifications of mitochondrial proteins regulate their function and activity, accordingly, impairment of ubiquitin proteasome system affects mitochondria homeostasis and organelle dynamics. In the present study we have investigated the role of the ubiquitin protease Ubp8 in S. cerevisiae respiration. We show that Ubp8 is necessary for respiration and its expression is upregulated in glycerol respiratory medium. In addition, we show that the respiratory defects in absence of Ubp8 are efficiently rescued by disruption of the E3 Ub-ligase Psh1, suggesting their epistatic link. Interestingly, we found also that Ubp8 is localized into mitochondria as single protein independently of SAGA complex assembly, thus suggesting an independent function from the nuclear one. We also show evidences on the importance of HAT Gcn5 in sustaining Ubp8 expression and affecting the amount of protein in mitochondria. Collectively, our results have investigated the role of Ubp8 in respiratory metabolism and highlight the role of ubiquitin related pathways in the mitochondrial functions of S. cerevisiae